Abstract
Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.
Highlights
Oligonucleotide therapeutics have gained in importance over the last decades as they can be utilized to interfere with almost every cellular process by selecting the appropriate sequence and format[1]
The sequence was originally selected as a putative antagomir against microRNA200c, which has been the subject of other research in our lab [23, 34,35]
The current study focuses on the stability and localization of the RNAs irrespective of their effector function and builds that basis for future sequence-specific degradation studies
Summary
Oligonucleotide therapeutics have gained in importance over the last decades as they can be utilized to interfere with almost every cellular process by selecting the appropriate sequence and format[1]. While the double-stranded representatives are mostly limited to RNA interference[5, 6], single-stranded oligonucleotides have a broader spectrum of applications. Compared to double-stranded RNAs, the single-stranded formats are more prone to nucleolytic degradation upon exposure to the cellular environment. Examples include ribose modifications in the 2’ position such as 2’-O-Methyl, 2’-Fluoro or locked nucleic acids (LNA) [18, 19]. Another highly nuclease protective intervention, especially in combination with the 2’-modifications mentioned above, is the replacement of the natural phosphodiester linkages by phosphorothioates where one of the non-bridging oxygens is replaced by sulfur[20]. The behavior of differently modified antagomirs in mice was investigated by Stoffel and coworkers[27] and an interesting study by Hirsch et al examined the duplex stability and localization of siRNA by intensity based FRET[28]
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